A Need for Self-Renewable Source of Mature Hepatocytes
The liver is the largest glandular organ in the body and performs multiple critical functions to keep the body pure of toxins and harmful substances. Liver function is mainly contributed by hepatocytes. Hepatocyte is the main cell type for glucose metabolism, lipid metabolism, urea metabolism and detoxification. It produces bile to digest fats; stores glucose, vitamins and iron; converts ammonia to urea and detoxifies the blood to rid it of harmful substances such as alcohol and drugs. In most liver diseases, hepatocytes are progressively loss to the extent where liver function fails and transplantation remains the gold standard of treatment. However, the medical complexity of the procedure coupled with a severe lack of healthy liver grafts creates an urgent need for more sustainable options. Worldwide, the mismatch between liver graft availability and patients waiting for a liver transplant has widened exponentially resulting in more patients dying while on the waiting list. This unmet clinical need has spurred much effort to develop cellular transplantation as an alternative to whole organ transplantation. Beside clinical needs, hepatocytes are constantly used in the industry for in vitro toxicology screens. Drug induced liver injury remains a major hurdle for many drugs in reaching clinical trials and in some cases drugs have been withdrawn from the shelf because of toxicity issues. This in part stems from the limitations of current in vitro hepatocyte toxicity models and mouse toxicity models to predict all forms of drug induced liver injury [1,2]. There is an urgent need for a renewable source of quality hepatocytes for both industrial and clinical applications. Hepatocytes isolated from fresh liver tissue lose their major drug metabolic enzyme Cytochrome p450 (CYP) activity after 24-48 hours of in vitro culture. Currently there is no method available for long term culture of primary hepatocyte, without the loss of their CYP drug metabolism functions. Besides the liver, a plausible renewable source of hepatocytes would be their derivation from pluripotent stem cells. However, these cells at best achieve functional activity of immature fetal hepatocytes which is much lower than mature adult hepatocytes [3]. In contrast, the liver stem cells described herein generate hepatocytes with adult hepatocyte features and the self-renewal capacity makes them a sustainable source of mature liver adult stem cells.
Structure and Cell Types in an Adult Liver
The liver lobule forms the functional basic unit of the liver [4]. Hepatocytes are compacted around a central vein and the portal triad (consisting of the bile ducts, hepatic vein and hepatic artery) are found at the edge of the lobule. While hepatocytes form the majority of the cells in the lobule, several other cells types are essential to form the ductal and vasculature networks, and immune surveillance of the organ. Other major cell types in the liver includes bile duct cells (cholangiocytes), liver sinusoidal endothelial cells, vasculature endothelial cells, immune cells including Pit cells, Kupffer cells and hepatic stellate cells. Within the liver lobules, hepatocytes are distributed to 3 different zones, determined by their proximity to the central vein or the portal triad located at the end of the lobule. Hepatocytes in the different zones are exposed to different niche environments and play specific functional roles in the liver. They can be distinguished by the expression of different markers, glucose and lipid metabolic functions.
Functional Hepatocytes and Cholangiocytes
Hepatocytes are the chief functional cells of the liver. As mentioned previously they perform an astonishing number of metabolic, endocrine and secretory functions. Roughly 80% of the mass of the liver is contributed by hepatocytes. In three dimensions, hepatocytes are arranged in plates that anastomose with one another. The cells are polygonal in shape with one or two prominent nucleoli and their sides can be in contact either with sinusoids (sinusoidal face) or neighboring hepatocytes (lateral faces). Hepatocyte function could be assessed by glucose storage, ion uptake, bile salt secretion, amino acid metabolic function, urea synthesis and drug metabolic function test.
The drug metabolizing function of hepatocytes is mediated by cytochrome P450s (CYPs). CYPs constitute the major enzyme family capable of catalyzing the oxidative biotransformation of most drugs. 90% of drugs are metabolized by six major CYPs (CYP3A4/5, CYP2C9, CYP2C19, CYP1A2, CYP2B6 and CYP2D6) [5]. CYP function can be enhanced by induction with specific drugs. CYP function and drug induction response is a major critical criterion used to assess the functional maturity of lab-made hepatocytes. While hepatocytes have been derived using various methods from embryonic and fetal tissues, the maturity and functionality of these hepatocytes as determined by the activities of the 6 major CYPs and their response to drug induction showed that they were functionally immature [6,13]. Therefore current methods known in the field of deriving hepatocytes do not solve the issue of obtaining large numbers of functionally mature hepatocytes for both industrial and clinical applications.
Besides hepatocytes, bile duct cells (cholangiocytes) are another type of liver parenchymal cell. Cholangiocytes are epithelial cells that line the intra- and extrahepatic ducts of the biliary tree [7]. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile. Typical cholangiocytes are polarized columnar cells. Their nucleoli are located near the basal membrane. Primary cilia extend from the cholangiocyte apical plasma membrane and into the ductal lumen. Microvilli formed by primary cilia on the apical plasma membrane significantly increase the cholangiocyte surface area. This enhances secretion and ion transport function of cholangiocytes [7]. Microvilli are a major structural characteristic used to assess the maturation of cholangiocytes. Currently, there are no reports of in vitro differentiated cholangiocytes with microvilli structures. Herein, we show that adult stem cells can generate mature cholangiocytes with microvilli.
Multiple Regenerative Cell Source in the Liver
In vivo, the liver is one of the most regenerative organs of the human body [8]. The human liver can lose up to ⅔ of its mass, maintains its critical functions and recover to the original mass within 8-15 days. Studies have shown during the recovery from a partial liver hepatectomy, hepatocytes from all regions of the liver proliferate [8,9], including bile duct cells. Recent lineage tracing experiments have identified proliferative hepatocytes in different regions of the liver, near the central vein [10] or the portal triad [11] during both normal liver homeostasis and liver injuries. Other than hepatocytes, potential proliferative cells have been identified in the bile duct regions that could replenish damage hepatocytes during injury [9,12].
Isolation of Adult Hepatic Stem Cells from the Adult Human Liver
The multi-cellular origin of liver stem cells suggests different methods can be used to isolate and derive stem cells from the liver. The ability to successfully isolate and expand liver stem cells and further differentiate them into functional hepatocytes for meaningful repopulation in an injured liver to deliver clinical benefit has become a top priority for liver stem cell biologists. To date, two groups have reported isolating stem cells from adult liver tissue that could be stably expanded in vitro for the long term. However, the hepatocytes derived from these stem cells are functionally immature and are unsuitable for use in clinical and industrial applications.
The first group (see WO2015/173425 A1) successfully isolated stem cells from liver tissue based on cells expressing 2 key markers, Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and Epithelial cell adhesion molecule (EPCAM). EPCAM expressing cells were embedded in matrigel to form a liver epithelial cell mass. The epithelial cell mass mainly consisted of cells expressing bile duct cell markers Keratin 19 (KRT19), Keratin 7 (KRT7) and EPCAM, suggesting that the origin of the cells was from bile ducts in the liver. While hepatocytes and cholangiocytes could be derived from this epithelial cell mass, no evidence was presented that the cells were mature. The hepatocytes were only shown to exhibit Cytochrome P450, Family 3, Subfamily A, Polypeptide 4 (CYP3A4) activity. This cytochrome activity is highly active in immature hepatocytes derived from embryonic or fetal tissues or stem cells [13]. There is no evidence that the cells exhibited mature Cytochrome P450, Family 2, Subfamily C, Polypeptide 9 (CYP2C9) functions, or that Cytochrome P450 (CYP) activity could be further induced with a drug. Similarly, derived cholangiocytes did not show mature phenotypes such as a polarized cell structure and the presence of cilia on the apical membrane. Thus, there is no evidence that the methods disclosed in WO2015/173425 were capable of generating mature hepatocytes and cholangiocytes.
A second group (see WO2014152321 A1) described isolating adult stem cells from various tissues or organs including stomach, small intestine, colon, intestinal metaplasia, fallopian tube (oviducts), kidney, pancreas, liver, and lung. Their isolated liver stem cell expressed mainly bile duct markers such as KRT7 and KRT19. The liver stem cells were shown to differentiate into Alpha-Fetoprotein (AFP) expressing hepatocytes. AFP is only found in embryonic and fetal immature hepatocytes. This suggested that these stem cells cannot generate fully mature hepatocytes. No metabolic functions were described, and no evidence was presented that the stem cells could differentiate into cholangiocytes. In addition, the in vivo transplantation data presented did not show that the liver stem cell could differentiate into functional hepatocytes in the mouse liver.